Piotrowska Justyna, Jodoi Yuki, Trang Nguyen Ha, Wawrzynska Anna, Takahashi Hideki, Sirko Agnieszka, Maruyama-Nakashita Akiko
Institute of Biochemistry and Biophysics Polish Academy of Sciences, ul. Pawinskiego 5A, 02-106 Warsaw, Poland.
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Fukuoka, Japan.
Plants (Basel). 2022 Oct 2;11(19):2595. doi: 10.3390/plants11192595.
Sulfur LIMitation1 (SLIM1) transcription factor coordinates gene expression in plants in response to sulfur deficiency (-S). SLIM1 belongs to the family of plant-specific EIL transcription factors with EIN3 and EIL1, which regulate the ethylene-responsive gene expression. The EIL domains consist of DNA binding and dimerization domains highly conserved among EIL family members, while the N- and C-terminal regions are structurally variable and postulated to have regulatory roles in this protein family, such that the EIN3 C-terminal region is essential for its ethylene-responsive activation. In this study, we focused on the roles of the SLIM1 C-terminal region. We examined the transactivation activity of the full-length and the truncated SLIM1 in yeast and . The full-length SLIM1 and the truncated form of SLIM1 with a deletion of C-terminal 106 amino acids (ΔC105) transactivated the reporter gene expression in yeast when they were fused to the GAL4 DNA binding domain, whereas the deletion of additional 15 amino acids to remove the C-terminal 120 amino acids (ΔC120) eliminated such an activity, identifying the necessity of that 15-amino-acid segment for transactivation. In the mutant, the transcript levels of sulfate transporter and the GFP expression derived from the promoter-GFP () transgene construct were restored under -S by introducing the full-length SLIM1, but not with the C-terminal truncated forms ΔC105 and ΔC57. Furthermore, the transcript levels of -S-responsive genes were restored concomitantly with an increase in glutathione accumulation in the complementing lines with the full-length SLIM1 but not with ΔC57. The C-terminal 57 amino acids of SLIM1 were also shown to be necessary for transactivation of a -S-inducible gene, , in a transient expression system using the promoter-GUS as a reporter. These findings suggest that the C-terminal region is essential for the SLIM1 activity.
硫限制1(SLIM1)转录因子可协调植物中基因的表达,以响应硫缺乏(-S)。SLIM1属于植物特异性EIL转录因子家族,与EIN3和EIL1一起调节乙烯响应基因的表达。EIL结构域由EIL家族成员中高度保守的DNA结合和二聚化结构域组成,而N端和C端区域在结构上是可变的,并推测在该蛋白家族中具有调节作用,例如EIN3的C端区域对其乙烯响应激活至关重要。在本研究中,我们重点关注了SLIM1 C端区域的作用。我们检测了全长和截短的SLIM1在酵母中的反式激活活性。当全长SLIM1和截短的C端缺失106个氨基酸的SLIM1(ΔC105)与GAL4 DNA结合结构域融合时,它们在酵母中可反式激活报告基因的表达,而额外缺失15个氨基酸以去除C端120个氨基酸(ΔC120)则消除了这种活性,这表明该15个氨基酸片段对于反式激活是必需的。在突变体中,通过导入全长SLIM1可在-S条件下恢复硫酸盐转运蛋白的转录水平以及源自启动子-GFP()转基因构建体的GFP表达,但截短形式的ΔC105和ΔC57则不能。此外,在全长SLIM1的互补系中,-S响应基因的转录水平随着谷胱甘肽积累的增加而恢复,但ΔC57则不然。在以启动子-GUS作为报告基因的瞬时表达系统中,SLIM1的C端57个氨基酸对于-S诱导基因的反式激活也是必需的。这些发现表明C端区域对于SLIM1的活性至关重要。